The long-range goal of this project is to understand how the mammalian neurogenic bHLH proteins MASH1 and ngn1 control the lineage-specific commitment and differentiation of multipotent neural progenitor cells, using the neural crest as a model system. MASH1 (more precisely hASH1) appears to play an important role in small-cell lung cancer (SCLC) and other neuroendocrine (NE) tumors. MASH1 and ngn1 may also be involved in tumors derived by malignant transformation of neural crest derivatives. This study will thus help in understanding the progression of these tumors. My proposal will focus on the identification and assessment of the lineage-specific cofactors for these bHLH proteins. Recent studies have suggested that two transcription factors, MEF2 and MyT1, serve as cofactors for MASH1 and ngn1 respectively. Two-hybrid selection will be used to isolate other cofactors. In this selection, modified bHLH domains of MASH1 and ngn1 will be used as baits to minimize potential background. The screening will be done with cDNA libraries from selected precursor lineages in which MASH1 and ngn1 function is normally required. Potential cofactors identified from the screening as well as MEF2 and MyT1 will be tested in gain-of-function and loss-of-function assays in primary cultures of neural crest or spinal cord cells. In vitro and in vivo experiments will demonstrate the physical interaction between the putative cofactors and the bHLH proteins. Biochemical assays will also determine whether the cofactor for MASH1 can enhance transcription form MASH1-dependent targets.